ObjectiveTo investigate the influence of connective tissue growth factor (CTGF) onthe collagen metabolism in human keloid fibroblasts with RNA interference (RNAi).Methods Human keloid fibroblasts (KFB) in vitro were transfected by 3 pairs of specific small interfering RNA (siRNA) CTGF plasmid synthesized for human CTGF,respectively.Reverse transcriptase-polymerase chain reaction (RT-PCR) contributed to the screening of the best siRNA in interfering of CTGF expression in human keloid fibroblasts to construct the plasmids,with the application of RNAi,to test the changes of expression level and collagen content of CTGF in transfected keloid fibroblasts through RT-PCR and Western blotting compared to its control groups.ResultsThe 3rd pair (C3) siRNA- CTGF expression of genes and proteins was remarkably inhibited after being interfered with human keloid fibroblasts,with inhibitory rates of 86.8 ％ and 65.6 ％.ConclusionsKeloid fibroblasts transfected by plasmid siRNA-CTGF effectively inhibite the expression of CTGF and deposition of collagen,and CTGF promotes the collagen synthesis in keloid development.

Objective To explore the use of rhomboid skin flap in expanded skin flap transfer. Methods A rhomboid skin flap was designed if the top soft part could not be fully utilized after expanded in a rotation skin flap. The flap pedicels were designed near the incision side. It should be ensured that ra-tio of the length to the width of the composite flap, which was composed of the rhomboid skin flap and the rotation skin flap, was 2.5∶1.0. Results Among these 11 patients with re-designed rhomboid skin flaps in the rotation skin flaps, the ratio of the length to the width reached to 3∶1 in some cases, but 2. 5∶1.0 in most cases. All the skin flaps survived, except one patient with disturbance of blood circulation in a small area and one with mild congestion. Conclusion The expanded soft tissue can be fully and rationally utilized to repair the skin defect in this design. Attention should be paid to the ratio of the length to the width of the composited flap, and it is better to select axial flap as the composite flap for safety. This method is safe, and worthy of recommendation.

Objective To investigate the operative methods and merits of nasal reconstruction in terms of the aesthetics. Methods The noses of 12 patients were reconstructed with an expanded triangle-shaped forehead flap with unilateral supratrochlear artery, a skin expander was placed obliquely under galea aponeurotica of forehead. Liquid was injected with conventional expansion method. Using the skin and the scars on nasal dorsum and tip as lining, based on intercanthal distance and aesthetic standard, a triangle-shaped forehead myocutaneous flap was designed over the expanded forehead skin tissue and used for a nasal reconstruction. The triangle-shaped flap was trimmed to different layer and reshaped based on aesthetic subunit.Results In twelve post-operative patients with nasal defect, no flap necrosis was found and the appearance of reconstructed noses were almost normal and satisfactory after follow-up for 6 months to 2 years. Conclusion The modified forehead myocutaneous flap according to aesthetic standard is safe and ideal for major nasal reconstruction. Meticulous moulding of triangle-shaped flap, nose interior with good blood supply, and primary insertion of nose stretcher are the key to a satisfactory appearance.

Objective To detect the characteristics and in vitro cell compatibility of human acellular dermal matrix (ADM) with the improved method.Methods Cell components of healthy human skins were removed by the improved method and the traditional method respectively.The porosity, degradation time in vitro of the ADM prepared by two methods and the cytotoxicity of the material infiltration liquid with the improved method on the adipose derived stem cells were detected.HE staining was used to detect the residual of the cells, the integrity of collagen and cell biocompatibility.Scanning electron microscopy (SEM) was used to detect the pore size.Results Both the two methods could completely remove the cells, and maintain the integrity of the collagen scaffold;The porosity of ADM with the improved method was higher (93.1±1.02)% than that of traditional method (74.27±2.04)% (P<0.05);There was no significant difference in the cytotoxicity and in vitro degradation time between the two kinds of ADM;While pore diameter of the improved method was significantly higher [(181.21±66.9) μm] than that [(102.38±15.63) μm] in dermal reticular surface with the traditonal method (P<0.05).Conclusions There is no obvious cytotoxicity of the ADM with the improved method, and therefore it is more suitable for cell adhesion growth with higher porosity and larger pore size.

Objective To explore the effects of recombinant plasmids of pGPU6/GFP/NeoshRNA-CTGF (shRNA-CTGF) on the type Ⅰ collagen (COL-Ⅰ) protein expression in keloid,through RNA interference on connective tissue growth factor (CTGF) in vivo and in vitro.Methods Recombinant plasmids were designed and constructed by specific shRNA-CTGF; After transfeced human keloid fibroblast with shRNA-CTGF in vitro,RT-PCR was used to detect the CTGF mRNA level,and Western blot to detect the secretion of COL-Ⅰ.After transfected the keloid of nude mice with shRNA-CTGF in vivo,RT-PCR was used to detect the CTGF and COL-Ⅰ mRNA level,and Western blot was used to detect the protein expression of COL-Ⅰ.Results Recombinant plasmids of CTGF were successfully constructed,and the CTGF gene expression was significantly decreased in vivo and in vitro by 86.8％ and 54.1 ％,respectively; Down-regulation of CTGF in vitro significantly inhibited the mRNA and protein level of COL-Ⅰ by 76.8％ and 65.6％,respectively; Down-regulation of CTGF in vivo significantly reduced the COL-Ⅰ mRNA and protein level by 52.7％ and 48.0％,respectively.Conclusions CTGF gene expression is successfully down-regulated by the recombinant plasmid of shRNA-CTGF in vivo and in vitro.shRNA-CTGF significantly reduces the COL-Ⅰ protein level in keloid.It implies that CTGF gene is a potential target in the therapy of pathological scar.

Objective To investigate the method of dermal-fat graft combined with secondary autologous fat transplantation in repairing severe facial depression and to evaluate the clinical effects.Methods Twelve cases of facial depression had been repaired by the transplantation of dermal-fat flap which was removed from the abdomen at the first stage.They were given fat granules injection 1 to 3 times postoperatively,and 3,6 and 3 patients were given fat granules injection three times,twice and once,respectively,with the interval period of 3 to 6 months.The result was based on comparison of the photos taken from preoperation and postoperation.Results All patients were healed primarily except one of which was formed hematoma after operation and scavenged thereafter.After 6 months to 2 years follow-up,all the patients had satisfactory facial contour.Conclusions Combined autologous fat granules with free dermal-fat graft to reconstruct severe facial depress is an easy,safe and effective technique and deserves to be recommended.

Objective To explore the possibility of primary culture of human adipose-derived stem cells in vitro and differentiation induction into epidermal cells.Methods Adipose-derived stem cells were primarily cultured by enzyme digestion method.The expression of CD29,CD34,CD44,CD49d,CD80 and CD106 was detected by immunohistochemical staining.ADSCs differention into epidermal cells in vitro were under induction medium.Proliferation activity and morphology of cells of induction group and control group were observed,and the expression of CKs in the two groups were also analyzed.Results ADSCs were successfully isolated and cultured from human liposuction tissue.It was revealed by immunofluorescence staining that ADSCs possessed specified expression of surface antigen of stem cells; ADSC successfully differentiated towards epidermal cells in vitro and possesed considerable high-level reproductive activity,cell morphology and expression of CKs also shown the tendency of differentiated into epidermal cells.Conclusions ADSCs can be isolated and cultured from human liposuction tissue,and can proliferate persistently.ADSCs possess specified expression of surface antigen.ADSCs can also be differentiated in vitro to the direction of epidermal cells.

Objective To investigate the specific silencing of connective tissue growth factor (CTGF) in a nude mouse keloid model,using RNA interference (RNAi) technique,and to provide the basis for gene therapy of keloid.Methods The nude mouse keloid model was established,and then transfected in vivo with well-amplifiating plasmid.The mRNA expression levels of CTGF mRNA and type Ⅰ collagen mRNA were detected with reverse transcription-polymerase chain reaction (RT-PCR).The distribution and protein expression levels of CTGF and type Ⅰ collagen were determined quantitatively using immunohistochemistry.Results The expression of CTGF at mRNA and protein levels was decreased in the experiment group,and the expression of type Ⅰ collagen at mRNA and protein levels was also decreased after transfection,as compared with negative control group and blank group,with significant difference between groups (P＜0.05).Moreover,the expression of type Ⅰ collagen and CTGF was positively correlated (r=0.979).Conclusions Keloid type Ⅰ collagen can be decreased through in vivo inhibiting CTGF expression.The transfection of CTGF gene in vivo may have effects on type Ⅰ collagen generation,and thus inhibit the keloid growth.

Objective To study the significance of HSP47 gene in keloid formation after in vivo study with RNAi technology and the recombinant HSP4 7 siRNA against heat shock protein 47 to keloid in a nude mice model.Methods We injected RNAi mixture into the keloid of a nude mice model in experimental group and PBS water(0.25 ml)into control group at the 16th days after establishing the models.After interference we observed the specimens and harvested specimens at 7th days for biochemical and pathological analysis.Results The expression of HSP47 mRNA reduced obviously and the collagen content also reduced in the experimental group.The rusults had statistical significance.Conclusion We can suppress the expression of HSP47 gene and then reduce the production of collagen after in vivo interfering experiment with HSP4 7 siRNA in keloid nude mice models using RNAi technique.This study cornfirms the mechanism that HSP47 promotes the keloid formation,which provides a new target to treat keloid.

Objective To explore the effect of epidermal growth factor (EGF) on tube formation of HUVEC induced by the secretion of angiogenesis factors of adipose-derived stem cells (ADSCs).Methods ADSCs were primarily cultured by enzyme digestion method.The flow cytomertry was performed to detect the expression of cell surface marker.ELISA was used to detect the expression of VEGF,HGF,and SDF-1 after given different doses of EGF.Tube formation assay was used to examine the effect of EGF on the tube formation induced by ADSCs.Results ADSCs were successfully isolated and cultured from human liposuction tissue and specific markers were expressed on ADSCs.EGF promoted the secretion of angiogenesis factors VEGF,HGF,and SDF-1,which were secreted by ADSCs.EGF pretreatment increased the ability of tube formation of HUVECs induced by ADSCs.Conclusions ADSCs induce the secretion of angiogenesis factors in vitro,and thus increase the ability of tube formation of HUVECs.EGF promotes the secretion ability of ADSCs,and the best concentration is 15 mg/L.